Methods and compositions related to physiologically responsive microneedle delivery systems

ABSTRACT

Disclosed herein are microneedle devices, kits comprising the microneedle devices, and methods of using the microneedle devices. Specifically, disclosed is a device for transport of a material across a biological barrier of a subject comprising: a plurality of microneedles each having a base end and a tip, with at least one pathway disposed at or between the base end and the tip; a substrate to which the base ends of the microneedles are attached or integrated; and at least one reservoir which is in connection with the base ends of the microneedles array, wherein the reservoir comprises an agent delivery system, wherein the agent delivery system comprises an agent to be transported across the biological barrier, or a means for producing an agent to be transported across the biological barrier, and a means for detecting a physiological signal from the recipient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of U.S. patent application Ser. No.15/999,770, filed Aug. 20, 2018, which was a U.S. National StageApplication of PCT US 2017/018319, filed Feb. 17, 2017, which claimspriority to U.S. Provisional Application No. 62/297,346, filed Feb. 19,2016, all of which are hereby incorporated by reference in theirentirety.

BACKGROUND OF THE INVENTION

Diabetes mellitus, as one of the most challenging chronic diseases,currently affects over 387 million people worldwide and this number isestimated to increase to around 500 million by 2030 (J. E. Shaw et al.Diabetes Research and Clinical Practice 2010, 87, 4; B. Belgium, IDFDiabetes Atlas, 6th edn, International Diabetes Federation 2013).Providing lifelong exogenous insulin is essential for the treatment oftype 1 diabetes (Matriano et al. Diabetes Care 2013, 36 Supply 1, S67;R. A. Hayward, Jama 1997, 278, 1663; D. R. Owens, B. Zinman, G. B.Bolli, The Lancet 2001, 358, 739; R. A. Hayward, Jama 1997, 278, 1663;D. R. Owens et al. The Lancet 2001, 358, 739). However, there was anestimated 4.9 million diabetes related deaths worldwide in 2014. A keyconstraint of the traditional insulin injection lies in inadequateglycemic control, which leads to diabetes complications, such asblindness, limb amputation and kidney failure. Conversely, overtreatmentwith insulin causes hypoglycemia, which can lead to behavioral andcognitive disturbance, seizure, brain damage, or death (Mo et al.Chemical Society Reviews 2014, 43, 3595; C. Ricordi et al. NatureReviews Immunology 2004, 4, 259; G. Steil, Advanced Drug DeliveryReviews 2004, 56, 125; Z. Gu et al. Chemical Society Reviews 2011, 40,3638).

Transplantation of insulin-producing cells has been intensively exploredfor treating type 1 diabetes (S. Schneider et al. Transplantation 2008,86, 1762). However, due to the host recognition of transplanted cells,dependence on donor cells and requirement of extensive immunosuppressivetherapy, direct cell implantation has a limited role in diabetes care(S. Merani et al. British Journal of Surgery 2008, 95, 1449; R.Nishimura et al. Transplantation Proceedings 2011, 43, 3239; A. R.Pepper et al. Nature Biotechnology 2015, 33, 518). An alternativetechnique is to encapsulate pancreatic β-cells in a semi-permeablecontainer, isolating and protecting them from the immune system whilestill allowing the diffusion and transportation of nutrients and oxygento the encapsulated cells (H. Zimmermann et al. Current Diabetes Reports2007, 7, 314; E. Pedraza et al., Proceedings of the National Academy ofSciences 2012, 109, 4245; C. C. Lin et al. Proceedings of the NationalAcademy of Sciences 2011, 108, 6380). Nevertheless, the cell-capsuleimplantation or withdrawal usually requires a surgical procedure. Moreimportantly, biocompatibility of the cell capsules is often compromisedresulting in persistent inflammation, formation of foreign body giantcells, fibrosis, damage to the surrounding tissues and failure of theimplant to control glucose (O. Veiseh, R. Langer, Nature 2015, 524, 39;Z. Gu, A. A. Aimetti, Q. Wang, T. T. Dang, Y. Zhang, O. Veiseh, H.Cheng, R. S. Langer, D. G. Anderson et al. ACS Nano 2013, 7, 4194; W.Tai et al. Biomacromolecules 2014, 15, 3495). What is needed in the artis a device and method for delivering compositions to a subject in needthereof, wherein the device and method do not require surgery.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show a schematic of the glucose responsive system (GRS)based on a microneedle-array patch integrated with pancreatic β-cellsand glucose signal amplifiers (GSA). FIG. 1A shows that without GSA,there is insignificant insulin release from the MN patch neither innormoglycemia nor hyperglycemia state. The MN patch is composed ofcrosslinked hyaluronic acid (grey). FIG. 1B shows that with GSA, thereis significant promoted insulin release triggered by a hyperglycemiastate. The MN patch is composed of crosslinked hyaluronic acid embeddingassembled layers of (from top to bottom) α-amylose and GSA.

FIGS. 2A-F show a characterization of a glucose signal amplifier (GSA).FIG. 2A shows TEM images of enzymes-encapsulated GSA pre-,post-incubated in 400 mg/dL glucose solution for 20 minutes, 2 hours and6 hours at 37° C. respectively. Scale bar is 200 nm. FIG. 2B (top) showsfluorescence 2.5D images of FITC-GA loaded GSA solution pre- andpost-incubated in 400 mg/dL glucose solution for 2 hours at 37° C. FIG.2B (bottom) shows distribution of the fluorescence intensity along theindicated white dash line. (a.u. represents “arbitrary unit”). FIG. 2Cshows the size distribution of GSA pre- and post-incubated in 400 mg/dLglucose solution for 6 hours. FIG. 2D shows phosphorescence lifetimeprofile for the GSA incubated in different glucose level solutionscontaining an oxygen concentration molecule probe. FIG. 2E showsphosphorescence lifetime profile for the GSA loaded with full or halfdose of GOx in 400 mg/dL glucose solutions. FIG. 2F shows the intensityof UV absorption at 330 nm of GSA in solutions with different glucoseconcentrations at 37° C. Error bars indicate standard deviation (s.d.)(n=3).

FIGS. 3A-I show in vitro glucose-responsive studies of GSA andcharacterization of the MN patch and L-S GRS. FIG. 3A shows in vitroaccumulated enzymes release profile of the GSA in solutions withdifferent glucose concentrations at 37° C. *P<0.05 for GSA in 400 mg/dLglucose solution compared with those in 100 or 0 mg/dL glucoseconcentration solutions. FIG. 3B shows glucose production from theα-amylose hydrolysis catalyzed by the released enzymes. *P<0.05 for GSAin 400 mg/dL glucose solution compared with those in 100 or 0 mg/dLglucose solutions. FIG. 3C shows secretion rate profile of L-S GRSsimulated by the inflow of different glucose solutions through amicrofluidics device (100 and 400 mg/dL). (n=3). FIG. 3D showsimmunofluorescence image of the pancreatic β-cell capsules stained withinsulin (green) and nucleus (blue). Scale bar is 500 μm. FIG. 3E (a-c)shows fluorescence images of the pancreatic β-cells from day 1 to day 3after the encapsulation. Cells were stained with calcium-AM (live,green) and ethidium homodimer (dead, red). Scale bar is 500 μm. (bottomright) The insulin secretion index of the cells capsules as the functionof time from day 1 to day 3 after encapsulation. Error bars indicates.d. (n=3). FIG. 3F shows a schematic of stimulated insulin secretionfrom the L-S GRS using a microfluidics device. KRB with differentglucose concentration flowed through the microfluidics channel andinsulin secreted by the pancreatic β-cell capsules was collected fromthe outlet. FIG. 3G shows pictures of the GSA-loaded MN patch. Scale baris 1 cm. FIG. 3H shows SEM image of the MN patch. Scale bar is 500 μm.FIG. 3I shows fluorescence microscopy image of the L-S GRS: MN patch wasloaded with rhodamine-labeled GSA and calcium AM-stained pancreaticβ-cell capsules were positioned on the back of the MN patch. Scale baris 500 μm.

FIGS. 4A-F show in vivo studies of L-S GRS for type 1 diabetestreatment. FIG. 4A shows mouse dorsum skin was transcutaneously treatedwith MN patches. Scale bar is 1 mm (top); H&E stained cross-section ofthe treated skin indicated by the area within black dashed line(bottom). The regions of skin muscles and fat tissues are labeled as Mand F, respectively. Scale bar is 200 μm. FIG. 4B shows in vivo studiesof the MN patches for STZ-induced type 1 diabetic mice treatment. Micewere subjected to transcutaneous administration with a variety of MNssamples: empty MNs without GRS (w/o GRS), MNs integrated with only L-GRS(L-GRS), MNs integrated with only S-GRS (S-GRS), MNs integrated withL-S-GRS (L-S GRS), MNs integrated with L-S-GRS but without GOx in S-GRS(L-S GRS (w/o GOx)), and MNs integrated with L-S-GRS but withoutα-amylose in S-GRS (L-S GRS (w/o AM)). *P<0.05 for administration withMN integrated with L-S GRS compared with the control groups. FIG. 4Cshows a change of BGLs of diabetic mice treated with additional MN (L-SGRS) 6 hours post administration. *P<0.05 for additional administrationwith MN compared with no additional administration. The black arrowsindicate the administration points. FIG. 4D shows a change of BGLs ofhealthy mice after the MN administration (MN L-S GRS or empty MN (MN w/oGRS)). Error bars indicate s.d. (n=5). FIG. 4E shows a tolerance testtoward diabetic mice 2 hours post administration of MNs with L-S GRS incomparison with the healthy control mice. The time points ofadministration were pointed out by the black arrows. FIG. 4F showsresponsiveness was calculated based on the area under the curve (AUC) in120 minutes, with the baseline set at the 0-minute blood glucosereading. Error bars indicate s.d. (n=5). *P<0.05 for diabetic micetreated with MN L-S GRS administration compared to the healthy mice.

FIG. 5 shows a glucose-responsive mechanism of hypoxia-sensitivehyaluronic acid (HS-HA).

FIG. 6 shows the TEM images and DLS measurements of GSA after incubationwith (from left to right) PBS buffer at 4° C., PBS buffer at 37° C. andPBS buffer containing a control level of glucose concentration (100mg/dL) at 37° C. for 6 hours, respectively.

FIG. 7 shows the normalized glucose production rate at different enzymesweight ratio between GA to AA. Error bars indicate s.d. (n=3).

FIG. 8 shows conversion of α-amylose to glucose in 10 mg/mL α-amylosesolution with 3 mg/mL AA and 6 mg/mL GA. Error bars indicate s.d. (n=3).

FIGS. 9A-B show CD spectra of (a) AA and (b) GA in the native states andthe ones released from the GSA incubated in solutions with 400 mg/dLglucose at 37° C. for 6 hours.

FIG. 10 shows glucose stimulated insulin secretion of the cells culturedon a 2D tissue culture plate and the cells encapsulated in 3D capsules30 hours post encapsulation. The amount of insulin secretion in 400mg/dL glucose KRB was normalized to insulin secretion in 100 mg/dLglucose KRB. Error bars indicate s.d. (n=3).

FIG. 11 shows the mechanical behavior of the GSA-loaded crosslinked MN.(n=5).

FIG. 12 shows BGLs of treated STZ-induced diabetic mice, which werecontinuously monitored in the first two hours after administration of MNw/o GRS, MN L-GRS, MN S-GRS, MN L-S-GRS, MN L-S GRS (w/o GOx) and MN L-SGRS (w/o AM). The black arrows indicate the administration points. Errorbars indicate s.d. (n=5). *P<0.05 for diabetic mice treated with MN L-SGRS administration compared to the healthy mice.

FIG. 13 shows a cytotoxicity study of GSA after 24-hour incubation withMIN6 cells. Error bars indicate s.d. (n=6).

FIG. 14A-B shows H&E-stained skin cross-section of surrounding tissueswith administration of PBS (a) or a MN patch (b) 2 days post-treatment.Those skin samples were at a distance of 5 mm from the MN injectionsite. Scale bar is 200 μm.

FIGS. 15A-B show rheological behavior of crosslinked m-HA hydrogelcontaining 98% complete cell growth medium at (a) 25° C. and (b) 37° C.using a TA Instruments AR-2000 stress controlled rheometer withsandpaper covered parallel-plate (25 mm). Experiments were performedwithin the linear viscoelastic regime at 0.5 Pa geometries with a 2 mmgap. Measurements were performed at least thrice to ensurereproducibility within ±10%.

SUMMARY OF THE INVENTION

Disclosed herein is a device for transport of a material across abiological barrier of a subject comprising: a plurality of microneedleseach having a base end and a tip, with at least one pathway disposed ator between the base end and the tip; a substrate to which the base endsof the microneedles are attached or integrated; at least one reservoirwhich is in connection with the base ends of the microneedles array,wherein the reservoir comprises an agent delivery system, wherein theagent delivery system comprises an agent to be transported across thebiological barrier, or a means for producing an agent to be transportedacross the biological barrier, and a means for detecting a physiologicalsignal from the recipient; and a signal amplifier system, wherein thesignal amplifier system comprises a component capable of amplifying thephysiological signal from the recipient.

Also disclosed herein is a device for transport of a material across abiological barrier of a subject comprising: a plurality of microneedleseach having a base end and a tip, with at least one pathway disposed ator between the base end and the tip; a substrate to which the base endsof the microneedles are attached or integrated; at least one reservoirwhich is in connection with the base ends of the microneedles array,wherein the reservoir comprises an agent delivery system, wherein theagent delivery system comprises an agent to be transported across thebiological barrier, or a means for producing an agent to be transportedacross the biological barrier, and a means for detecting a physiologicalsignal, wherein the agent delivery system comprises a feedbackcomponent, such that volume or amount of the agent to be transportedacross the biological barrier can be altered based on the physiologicalsignal.

Further disclosed herein is a method of treating a disease in a subjectin need thereof, the method comprising: a) providing a microneedle patchto the subject, wherein the microneedle patch comprises: i) a pluralityof microneedles each having a base end and a tip; ii) a substrate towhich the base ends of the microneedles are attached or integrated; iii)at least one reservoir which is in connection with the base ends of themicroneedles array, wherein the reservoir comprises an agent deliverysystem, wherein the agent delivery system comprises an agent to betransported across the biological barrier, or a means for producing anagent to be transported across the biological barrier, and a means fordetecting a physiological signal from the recipient; and iv) a signalamplifier system, wherein the signal amplifier system comprises acomponent capable of amplifying the physiological signal from therecipient; b) inserting the microneedles into the biological barrier;and c) delivering the agent through the microneedles and into thebiological barrier, wherein the amount or volume of agent delivered isdetermined by a signal received from the signal amplifier system.

Also disclosed herein is a method of treating a disease in a subject inneed thereof, the method comprising: a) providing a microneedle patch tothe subject, wherein the microneedle patch comprises: i) a plurality ofmicroneedles each having a base end and a tip; ii) a substrate to whichthe base ends of the microneedles are attached or integrated; iii) atleast one reservoir which is in connection with the base ends of themicroneedles array, wherein the reservoir comprises an agent deliverysystem, wherein the agent delivery system comprises an agent to betransported across the biological barrier, or a means for producing anagent to be transported across the biological barrier, and a means fordetecting a physiological signal, wherein the agent delivery systemcomprises a feedback component, such that volume or amount of the agentto be transported across the biological barrier can be altered based onthe physiological signal; and b) inserting the microneedles into thebiological barrier; and c) delivering the agent through the microneedlesand into the biological barrier, wherein the amount or volume of agentdelivered is based on the physiological signal.

Disclosed herein is a kit of parts for delivering a therapeutic,prophylactic or diagnostic agent across a biological barrier comprising:a) a microneedle patch comprising a plurality of microneedles eachhaving a base end and a tip, and a substrate to which the base ends ofthe microneedles are attached or integrated; b) at least one reservoirwhich is in connection with the base ends of the microneedles array,wherein the reservoir comprises an agent delivery system, wherein theagent delivery system comprises an agent to be transported across thebiological barrier, or a means for producing an agent to be transportedacross the biological barrier, and a means for detecting a physiologicalsignal from the recipient; and c) a signal amplifier system, wherein thevolume or amount of agent to be transported is altered based on a signalreceived from the signal amplifier system.

Also disclosed herein is a kit of parts for delivering a therapeutic,prophylactic or diagnostic agent across a biological barrier comprising:a) a microneedle patch comprising a plurality of microneedles eachhaving a base end and a tip, and a substrate to which the base ends ofthe microneedles are attached or integrated; b) at least one reservoirwhich is in connection with the base ends of the microneedles array,wherein the reservoir comprises an agent delivery system, wherein theagent delivery system comprises an agent to be transported across thebiological barrier, or a means for producing an agent to be transportedacross the biological barrier, and a means for detecting a physiologicalsignal from the recipient.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Terms used throughout this application are to be construed with ordinaryand typical meaning to those of ordinary skill in the art. However,Applicant desires that the following terms be given the particulardefinition as defined below.

As used in the specification and claims, the singular form “a,” “an,”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

The terms “about” and “approximately” are defined as being “close to” asunderstood by one of ordinary skill in the art. In one non-limitingembodiment the terms are defined to be within 10%. In anothernon-limiting embodiment, the terms are defined to be within 5%. In stillanother non-limiting embodiment, the terms are defined to be within 1%.

“Activities” of a protein, including those relating to “bioactivity,”include, for example, transcription, translation, intracellulartranslocation, secretion, phosphorylation by kinases, cleavage byproteases, and/or homophilic and heterophilic binding to other proteins.

“Biocompatible” generally refers to a material and any metabolites ordegradation products thereof that are generally non-toxic to therecipient and do not cause any significant adverse effects to thesubject.

A “composition” is intended to include a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label) or active, such as an adjuvant.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. “Consisting of” shall mean excluding morethan trace elements of other ingredients and substantial method stepsfor administering the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

A “control” is an alternative subject or sample used in an experimentfor comparison purpose. A control can be “positive” or “negative.”

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations, applications or dosages.

As used herein, the term “high glucose conditions” refers to anenvironment having a glucose concentration greater than or equal to 200mg/dL. For example, “high blood glucose levels” refer to glucose levelsin the blood stream greater than or equal to 200 mg/dL. In someembodiments, high glucose conditions are 200-400 mg/dL. In otherembodiments, high glucose conditions are 300-400 mg/dL.

As used herein, the term “low glucose conditions” refers to anenvironment having a glucose concentration from 0 to 200 mg/dL. Forexample, “low blood glucose levels” refer to glucose levels in the bloodstream less than 200 mg/dL.

The terms “peptide,” “protein,” and “polypeptide” are usedinterchangeably to refer to a natural or synthetic molecule comprisingtwo or more amino acids linked by the carboxyl group of one amino acidto the alpha amino group of another.

The term “carrier” or “pharmaceutically acceptable carrier” means acarrier or excipient that is useful in preparing a pharmaceutical ortherapeutic composition that is generally safe and non-toxic, andincludes a carrier that is acceptable for veterinary and/or humanpharmaceutical or therapeutic use. As used herein, the terms “carrier”or “pharmaceutically acceptable carrier” encompasses can includephosphate buffered saline solution, water, emulsions (such as anoil/water or water/oil emulsion) and/or various types of wetting agents.As used herein, the term “carrier” encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, orother material well known in the art for use in pharmaceuticalformulations and as described further below.

As used herein, the term “polymer” refers to a relatively high molecularweight organic compound, natural or synthetic, whose structure can berepresented by a repeated small unit, the monomer (e.g., polyethylene,rubber, cellulose). Synthetic polymers are typically formed by additionor condensation polymerization of monomers. As used herein, the term“copolymer” refers to a polymer formed from two or more differentrepeating units (monomer residues). By way of example and withoutlimitation, a copolymer can be an alternating copolymer, a randomcopolymer, a block copolymer, or a graft copolymer. It is alsocontemplated that, in certain aspects, various block segments of a blockcopolymer can themselves comprise copolymers.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed.

The terms “therapeutically effective amount” or “therapeuticallyeffective dose” refer to the amount of a composition, such asglucose-modified insulin bound to a glucose-binding structure, that willelicit the biological or medical response of a tissue, system, animal,or human that is being sought by the researcher, veterinarian, medicaldoctor or other clinician over a generalized period of time. In someembodiments, a desired response is the control of type I diabetes. Insome instances, a desired biological or medical response is achievedfollowing administration of multiple dosages of the composition to thesubject over a period of days, weeks, or years.

The term “subject” or “recipient” is defined herein to include animalssuch as mammals, including, but not limited to, primates (e.g., humans),cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and thelike. In some embodiments, the subject is a human.

The terms “treat,” “treating,” “treatment,” and grammatical variationsthereof as used herein, include partially or completely delaying,alleviating, mitigating or reducing the intensity of one or moreattendant symptoms of a disorder or condition and/or alleviating,mitigating or impeding one or more causes of a disorder or condition.Treatments according to the invention may be applied preventively,prophylactically, pallatively or remedially.

In some instances, the terms “treat”, “treating,” “treatment” andgrammatical variations thereof, include controlling blood sugar levelsand reducing the severity of type I diabetes symptoms as compared withprior to treatment of the subject or as compared with the incidence ofsuch symptom in a general or study population.

The term “type I diabetes” refers to the form of diabetes mellitusresulting from the autoimmune destruction of insulin-producing cells andreduction of the body's ability to produce insulin. The loss of insulinresults in increased blood sugar.

DETAILED DESCRIPTION

Disclosed herein is a device for transport of a material across abiological barrier of a subject comprising:

a plurality of microneedles each having a base end and a tip, with atleast one pathway disposed at or between the base end and the tip;

a substrate to which the base ends of the microneedles are attached orintegrated; and

at least one reservoir which is in connection with the base ends of themicroneedles array, wherein the reservoir comprises an agent deliverysystem, wherein the agent delivery system comprises an agent to betransported across the biological barrier, or a means for producing anagent to be transported across the biological barrier, and a means fordetecting a physiological signal from the recipient.

The agent delivery system can, for example, comprise a feedbackcomponent, such that volume or amount of the agent to be transportedacross the biological barrier can be altered based on the physiologicalsignal. The feedback component can comprise an “on and off” switch, suchthat when a signal is detected, the agent delivery system can deliver anagent to the recipient, but when no signal is detected, no agent isdelivered. Conversely, the detection of a signal can have the oppositeeffect, wherein the agent delivery system defaults to delivery of anagent to the recipient, unless a signal is detected, which causes theagent delivery system not to release an agent for delivery to therecipient. By way of illustration, the feedback component can detect thepresence of a pathogen in the recipient (the agent), and when the agentis detected, the feedback component can allow for the release of anantibody from the reservoir.

In another example, the feedback component can detect changes in aphysiological signal, such as pH or temperature. The feedback componentcan comprise a “cut off value” such that when the pH or the temperaturechanges by a certain amount, or reaches a certain numerical value (a pHbelow 6.5, for example, or a temperature above 99.1, for example), thefeedback component allows for a change in the release of the agent, orthe amount of the agent released, and subsequently administered to therecipient.

The feedback component can also adjust the amount or volume of the agentreleased based on the amount of signal detected, so that a greateramount of signal detected can result in a greater amount of agentreleased, or conversely, a greater amount of signal detected can resultin a smaller amount of agent released.

Optionally, the device can comprise a signal amplifier system, whereinthe signal amplifier system comprises a component capable of amplifyingthe physiological signal from the recipient. The signal amplifier systemworks by increasing the signal, which increases the signal detected bythe agent delivery system. This can be done in the case where there is aneed to detect very small changes in the physiological signal, or whenthe agent delivery system is not sensitive enough to detect a changewithout amplification of the signal. An example of a signal amplifiersystem can be found in Example 1.

The signal amplifier system can be integrated within the microneedles.For example, the signal amplifier system can be in the tips of themicroneedles, either as a coating on the outside of the microneedle, orinside the tips of the microneedle.

The physiological signal detected can, in one aspect, be any substancepresent in the recipient. For example, the physiological signal can be abiological substance or a drug. The substance can either occur naturallyin the recipient, or can be a non-endogenous, or foreign, substance. Inanother aspect, the physiological response in the subject can comprisephysiological environment factors, including pH and temperature.Examples of physiological signals include, but are not limited to,glucose, cholesterol, bilirubin, creatine, metabolic enzymes,hemoglobin, heparin, clotting factors, uric acid, carcinoembryonicantigen or other tumor antigens, reproductive hormones, oxygen, pH,temperature, alcohol, tobacco metabolites, and illegal drugs.

The agent in the reservoir to be delivered to the recipient can be atherapeutic, prophylactic, or diagnostic agent. For example, the agentcan be selected from the group consisting of peptides, proteins,carbohydrates, nucleic acid molecules, lipids, organic molecules,biologically active inorganic molecules, and combinations thereof. Forexample, a wide range of drugs may be formulated for delivery with thepresent microneedle devices and methods. As used herein, the terms“drug” or “drug formulation” are used broadly to refer to anyprophylactic, therapeutic, or diagnostic agent, or other substance thatwhich may be suitable for introduction to biological tissues, includingpharmaceutical excipients and substances for tattooing, cosmetics, andthe like. The drug can be a substance having biological activity. Thedrug formulation may include various forms, such as liquid solutions,gels, solid particles (e.g., microparticles, nanoparticles), orcombinations thereof. The drug may comprise small molecules, large(i.e., macro-) molecules, or a combination thereof. In representative,not non-limiting, embodiments, the drug can be selected from among aminoacids, vaccines, antiviral agents, gene delivery vectors, interleukininhibitors, immunomodulators, neurotropic factors, neuroprotectiveagents, antineoplastic agents, chemotherapeutic agents, polysaccharides,anti-coagulants, antibiotics, analgesic agents, anesthetics,antihistamines, anti-inflammatory agents, and viruses.

The drug may be selected from suitable proteins, peptides and fragmentsthereof, which can be naturally occurring, synthesized or recombinantlyproduced. In one embodiment, the drug formulation includes insulin.

The drug formulation may further include one or more pharmaceuticallyacceptable excipients, including pH modifiers, viscosity modifiers,diluents, etc., which are known in the art. Specifically, the agent canbe insulin.

The device disclosed herein can comprise the agent for release itself,or a means for producing an agent to be transported across thebiological barrier reservoir. One example of a means for producing anagent is cells. The cells can be mammalian cells, such as human cells,or can be cells from any other source, which are capable of producing anagent for administration to a recipient. For example, the cells canpancreatic β cells or stem cell-differentiated human pancreatic cells.

The agent, or the means for producing the agent, can be disposed in areservoir which is semi-permeable, for example. This can allow for theexchange of fluid with the recipient, such that the feedback componentcan be in fluid communication with the recipient, and thereby detectchanges in physiological signal of the recipient. For example, thereservoir can comprise cells, wherein the cells are sensitive to changesin a physiological signal from the recipient. Such physiological changesin the recipient can stimulate the cells to release an agent, or to stopreleasing an agent, as described above in regard to the feedbackcomponent. In one example, the reservoir can comprise an alginatemicrogel.

In one example, the signal amplifier system comprises a glucose signalamplifier (referred to as a “GSA” herein). The signal amplifier systemcan comprise self-assembled polymeric nanosized vesicles. By way ofspecific example, the glucose signal amplifier can comprise glucoseoxidase, α-amylase, and glucoamylase.

In regard to the microneedles themselves, they can be constructed from avariety of materials, including metals, ceramics, semiconductors,organics, polymers, and composites. Preferred materials of constructioninclude pharmaceutical grade stainless steel, gold, titanium, nickel,iron, tin, chromium, copper, palladium, platinum, alloys of these orother metals, silicon, silicon dioxide, and polymers. Representativebiodegradable polymers include polymers of hydroxy acids such as lacticacid and glycolic acid polylactide, polyglycolide,polylactide-co-glycolide, and copolymers with PEG, polyanhydrides,poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valericacid), and poly(lactide-co-caprolactone). Representativenon-biodegradable polymers include polycarbonate, polyester, andpolyacrylamides.

The microneedles should have the mechanical strength to remain intactwhile being inserted into the biological barrier, while remaining inplace for up to a number of days, and while being removed. Inembodiments where the microneedles are formed of biodegradable polymers,the microneedle must to remain intact at least long enough for themicroneedle to serve its intended purpose (e.g., its conduit functionfor delivery of drug). The microneedles should be sterilizable usingstandard methods such as ethylene oxide or gamma irradiation.

The microneedles can have straight or tapered shafts. In a preferredembodiment, the diameter of the microneedle is greatest at the base endof the microneedle and tapers to a point at the end distal the base. Themicroneedle can also be fabricated to have a shaft that includes both astraight (untapered) portion and a tapered portion. The needles may alsonot have a tapered end at all, i.e. they may simply be cylinders withblunt or flat tips. A hollow microneedle that has a substantiallyuniform diameter, but which does not taper to a point, is referred toherein as a “microtube.” As used herein, the term “microneedle” includesboth microtubes and tapered needles unless otherwise indicated.

The microneedles can be oriented perpendicular or at an angle to thesubstrate. Preferably, the microneedles are oriented perpendicular tothe substrate so that a larger density of microneedles per unit area ofsubstrate can be provided. An array of microneedles can include amixture of microneedle orientations, heights, or other parameters.

The microneedles can be formed with shafts that have a circularcross-section in the perpendicular, or the cross-section can benon-circular. For example, the cross-section of the microneedle can bepolygonal (e.g. star-shaped, square, triangular), oblong, or anothershape. The cross-sectional dimensions can be between about 1 μm and 1000μm, such that the base can be about 200-600 μm, and the tip can bebetween 1 and 20 μm. In one embodiment, the microneedle can beapproximately 400 μm at the base, and approximately 5 μm at the tip.

The length of the microneedles typically is between about 10 μm and 1mm, preferably between 400 μm and 1 mm. For example, the length of themicroneedle can be approximately 800 μm. The length is selected for theparticular application, accounting for both an inserted and uninsertedportion. An array of microneedles can include a mixture of microneedleshaving, for example, various lengths, outer diameters, inner diameters,cross-sectional shapes, and spacings between the microneedles.

The reservoir can be connected to the tip of the microneedle, such thatan agent stored or produced in the reservoir can flow from the reservoirand out through the microneedle tip, into the target tissue. Thereservoir is to provide suitable, leak-free storage of the agent, or themeans of producing the agent, before it is to be delivered.

The reservoir of a single microneedle device can include a plurality ofcompartments that are isolated from one another and/or from a portion ofthe microneedles in an array. The device can, for example, be providedto deliver different agents through different needles, or to deliver thesame or different agents at different rates or at different times.Alternatively, the contents of the different compartments can becombined with one another, for example, by piercing, or otherwiseremoving, a barrier between the compartments, so as to allow thematerials to mix.

The microneedle and substrate are made by methods known to those skilledin the art. Examples include microfabrication processes, by creatingsmall mechanical structures in silicon, metal, polymer, and othermaterials. Three-dimensional arrays of hollow microneedles can befabricated, for example, using combinations of dry etching processes;micromold creation in lithographically-defined polymers and selectivesidewall electroplating; or direct micromolding techniques using epoxymold transfers. These methods are described, for example, in U.S. Ser.No. 09/095,221, filed Jun. 10, 1998; U.S. Ser. No. 09/316,229, filed May21, 1999; Henry, et al., “Micromachined Needles for the TransdermalDelivery of Drugs,” Micro Electro Mechanical Systems, Heidelberg,Germany, pp. 494-98 (Jan. 26-29, 1998).

Also disclosed herein are methods of treating a disease in a subject inneed thereof, the method comprising:

a) providing a microneedle patch to the subject, wherein the microneedlepatch comprises:

-   -   i) a plurality of microneedles each having a base end and a tip;    -   ii) a substrate to which the base ends of the microneedles are        attached or integrated;    -   iii) at least one reservoir which is in connection with the base        ends of the microneedles array, wherein the reservoir comprises        an agent delivery system, wherein the agent delivery system        comprises an agent to be transported across the biological        barrier, or a means for producing an agent to be transported        across the biological barrier, and a means for detecting a        physiological signal from the recipient; and;    -   b) inserting the microneedles into the biological barrier; and    -   c) delivering the agent through the microneedles and into the        biological barrier, wherein the amount or volume of agent        delivered is determined by a signal received from the signal        amplifier system.

As discussed above, the agent delivery system can, for example, comprisea feedback component, such that volume or amount of the agent to betransported across the biological barrier can be altered based on thephysiological signal. The feedback component can essentially comprise an“on and off” switch, such that when a signal is detected, the agentdelivery system can deliver an agent to the recipient. Conversely, thedetection of a signal can have the opposite effect, thereby causing theagent delivery system not to release an agent for delivery to therecipient. By way of illustration, the feedback component can detect thepresence of a pathogen in the recipient, and when the analyte isdetected, the feedback component can allow for the release of anantibody from the reservoir.

Also discussed above, the device can comprise a signal amplifier system,wherein the signal amplifier system comprises a component capable ofamplifying the physiological signal from the recipient. The signalamplifier system works by increasing the signal, which increases thesignal detected by the agent delivery system. This can be done in thecase where there is a need to detect very small changes in thephysiological signal, or when the agent delivery system is not sensitiveenough to detect a change without amplification of the signal. Anexample of a signal amplifier system can be found in Example 1.

Also disclosed herein are kits. The kits can include parts for use withthe methods disclosed herein. For example, the kits can comprise theparts needed to form the devices disclosed herein. Disclosed is a kit ofparts for delivering a therapeutic, prophylactic or diagnostic agentacross a biological barrier comprising: a) a microneedle patchcomprising a plurality of microneedles each having a base end and a tip,and a substrate to which the base ends of the microneedles are attachedor integrated; b) at least one reservoir which is in connection with thebase ends of the microneedles array, wherein the reservoir comprises anagent delivery system, wherein the agent delivery system comprises anagent to be transported across the biological barrier, or a means forproducing an agent to be transported across the biological barrier, anda means for detecting a physiological signal from the recipient. Theagent delivery system can further comprise a feedback component. Alsodisclosed as part of the kit can be a signal amplifier system, which isdiscussed herein. The kit can also comprise written instructions foruse.

EXAMPLES Example 1: Microneedle Patch Platform

Disclosed herein is a painless microneedle (MN) patch platform tomodulate the insulin secretion from pancreatic β-cells forglucose-responsive regulation of blood glucose levels (BGLs) withoutimplantation. As shown in FIG. 1, this strategy integrates both live(cell-based) and synthetic glucose-responsive systems (L-S GRS) to allowthe externally positioned β-cell capsules to sense glucose signals andto secrete insulin through the MN in a minimally invasive manner. Theinitial design integrated cell capsules with the MN patch made from thecrosslinked hyaluronic acid (HA) (FIG. 1a ). It was expected that undera hyperglycemic state, glucose could diffuse through the MN and interactwith β-cells encapsulated in the alginate microgels in order to promoteinsulin secretion. However, due to the limited diffusion of glucose, thepatch did not effectively respond to a hyperglycemic state and aninsignificant increase in insulin secretion was detected. To effectivelytrigger the cellular response, the MN matrix reported here specificallycontains synthetic “glucose-signal amplifiers” (GSAs) (FIG. 1b ). Thisinnovative GSA is featured with self-assembled polymeric nanosizedvesicles entrapping three enzymes: glucose oxidase (GOx), α-amylase (AM)and glucoamylase (GA). GOx converts glucose into gluconic acid in thepresence of oxygen. AM hydrolyses the α-amylose into disaccharides andtrisaccharides, which further converts to glucose by GA (N. Gurung etal. Process Biochemistry 2003, 38, 1599; L. Kandra, Journal of MolecularStructure: THEOCHEM 2003, 666-667, 487).

Once subjected to the elevated BGLs, the GSA comprised ofhypoxia-sensitive materials quickly disassociates to release theencapsulated enzymes in response to the rapid glucose oxidation by GOxand oxygen consumption (J. Yu et al. Proceedings of the National Academyof Sciences 2015, 112, 8260; O. Veiseh et al. Nature 2015, 524, 39):

The released enzymes subsequently hydrolyze α-amylose (S. Peat, et al.Nature 1953, 172, 158; J. F. Robyt, D. French, Archives of Biochemistryand Biophysics 1967, 122, 8; W. J. Whelanet al. Nature 1952, 170, 748)embedded in the MN matrix, generating a local glucose-concentrated site.The “amplified” glucose effectively diffuses into the externallypositioned β-cell capsules, promoting secretion and diffusion of insulininto the vascular and lymph capillary networks (A. J. Harvey et al.Pharmaceutical Research 2010, 28, 107). Using streptozotocin(STZ)-induced type 1 diabetic mouse as an animal model, it wasdemonstrated that the GRS consisting of ˜10⁷ β-cells quickly respondedto a hyperglycemic state, declined and maintained BGLs at a reducedlevel for up to 10 hours. This cellular-synthetic hybridglucose-responsive device with a physiological-signal amplifier modalitypresents a useful alternative to pancreatic β-cells implantation fortight regulation of BGLs.

GSA was prepared by the solvent dialysis method for encapsulating threeenzymes (J. E. Chung et al. Nature Nanotechnology 2014, 9, 907; H. Yu etal. Nature Communications 2014, 5). Briefly, amine-functionalized2-nitroimidazole (NI) groups were covalently conjugated to the HA via anamide bond. The hypoxia-sensitive HA (HS-HA) functionalized withhydrophobic NI groups readily self-assembled into GSAs in the aqueoussolution containing GOx, α-amylase and amyloglucosidase (FIG. 5). Undera hypoxic condition, the hydrophobic NI groups were reduced tohydrophilic 2-aminomidazoles via a single-electron reaction with NADPHcatalyzed by nitroreductases (Y. Seki et al. Journal of biochemistry1970, 67, 389). The reduced product with amine groups was water-soluble,which facilitated the disassembly of GSA (J. Yu et al. Proceedings ofthe National Academy of Sciences 2015, 112, 8260; R. J. Hickey et al.Journal of the American Chemical Society 2011, 133, 1517). Thetransmission electron microscopy (TEM) image (FIG. 2a ) showed that theGSA had a spherical shape with a monodisperse size. The averagehydrodynamic size of GSA measured by dynamic light scattering (DLS) was340 nm (FIG. 2c ), which was consistent with the TEM images. Thezeta-potential of GSA was determined as −45.7±2.4 mV due to the residualcarboxyl of HA. The fluorescence image of GSA with fluoresceinisothiocyanate (FITC)-labeled GA and AM further verified successfulco-encapsulation of the enzymes (FIG. 2b ). The loading capacity of GSAbased on all the enzymes was determined as 7.4±0.5 wt % and loadingefficiency as 16.1±1.0 wt %. The GSA was stable when incubated at 4° C.and no noticeable turbidity change was observed over two weeks.

To assess the glucose-responsive capability of GSA in vitro, thevesicles were examined in 1×PBS buffer solutions with various glucoseconcentrations, including a typical hyperglycemic level (400 mg/dL), anormoglycemia level (100 mg/dL), and a control level (0 mg/dL). Thehyperglycemia level generated a relatively lower oxygen environment inthe GSA compared to the other two control groups, which was verified byan oxygen-sensitive phosphorescent molecular probe (FIG. 2d ). Theoxygen level inside the GSA gradually reduced over time and reachedequilibrium within 20 min. The oxygen consumption kinetics could befurther modulated by altering the amount of GOx loaded into the vesicle,which showed a clearly delayed hypoxic effect with a half dose of GOx(FIG. 2e ). With the decline of oxygen level, the NI groups wereeffectively reduced by NADPH added into the solution. Correspondingly,the characteristic peak of NI at 330 nm in UV-Vis spectra decreasedrapidly, which substantiated this bio-reduction reaction (FIG. 2f ). Dueto the generation of water-soluble pendant groups on HS-HA, the GSAbegan to dissociate and subsequently release the encapsulated enzymes.As shown in TEM images, the GSA in 400 mg/dL glucose solutionexperienced gradual morphology changes from 20 min to 6 h (FIG. 2a ),which was consistent with the remarkable decline in the averagehydrodynamic size, indicated by DLS (FIG. 2c ). In contrast, GSAincubated with no glucose or 100 mg/dL glucose displayed stablehydrodynamic size and no noticeable morphology change (FIG. 6).Furthermore, the release of encapsulated FITC-labeled enzymes from thedissociated vesicles was visualized by fluorescence microscopy. Thefluorescence signal intensity was significantly decreased and presentedhomogeneous distribution after 2 hours, suggesting that the enzymesescaped from the dissociated GSA and evenly dispersed in the solution(FIG. 2b ).

The enzyme release kinetics in response to the glucose level changeswere next examined No significant amount of released enzymes from GSAwas detected within 24 h of incubation at a normal glucose level (100mg/dL) and a control level (0 mg/dL) (FIG. 3a ). In sharp contrast, arapid enzyme release rate was achieved from the GSA in the first 2 hoursat a hyperglycemic environment (400 mg/dL). This was attributed to thefaster reduction of NI groups, which was induced by the hypoxiccondition upon glucose oxidation.

Afterwards, the conversion from α-amylose to glucose catalyzed by thereleased enzymes from GSA was further investigated. The encapsulationratio of AA to GA was pre-optimized as 1:2 by analyzing their enzymatichydrolysis capability of α-amylose, indicated by the glucose productionrate (FIG. 7). When AA and GA were utilized to saccharify 10 mg/mLα-amylose solution sequentially, the glucose production was readilyincreased to 816±26 mg/dL, yielding an 81.6% conversion rate ofα-amylose (FIG. 8). The circulation dichroism (CD) spectra confirmedthat the released enzymes GA and AA from GSA maintained their secondaryconformational structures (FIG. 9). Meanwhile, when the GSA wasincubated in α-amylose solutions with various glucose concentrations, asignificantly faster glucose production was achieved when incubated with400 mg/dL glucose compared to the one with 100 mg/dL glucose (FIG. 3b ).It indicated that the enzymatic hydrolysis of α-amylose was activated bythe gradual release of enzymes associated with the disassembly of GSA.Taken together, once “sensing” the elevated glucose level, the GSA couldbe activated to release the enzymes, which promoted theα-amylose-to-glucose conversion to amplify the glucose signal fordownstream action.

The use of MN patches for the delivery of insulin from pancreatic β-cellcapsules was further investigated. To create the “live”glucose-responsive component of the L-S GRS, the mouse islets β-celllines were encapsulated in alginate microgels with RGD (C. C. Lin et al.Proceedings of the National Academy of Sciences 2011, 108, 6380.) andtype IV collagen (L. M. Weber et al. Biomaterials 2007, 28, 3004.) at2×10⁶ cell/mL packing density to provide a benign environment withbiomimetic cell-ECM adhesive interactions. Successful encapsulation wasvisualized by fluorescence microscopy with the concentrated cells andhomogenous distribution of the secreted insulin surrounding the capsules(FIG. 3d ). The size of the obtained capsule was 735±27 μm. The glucosestimulated insulin secretion (GSIS) analysis and live-dead assay wereperformed after day 1 to day 3 to validate that the encapsulated β-cellsmaintained their viability and functionality (FIG. 3e ) C. C. Lin et al.Proceedings of the National Academy of Sciences 2011, 108, 6380.). Theresults indicated that the encapsulated β-cells could survive for arelatively long period of time and maintain normal glucose-responsiveinsulin secretion capability when compared their insulin secretion indexwith cells cultured on a 2D tissue culture plate (FIG. 10).

Meanwhile, the MN patch was fabricated using a micromolding approach.The resulting MN device had 400 pyramid needles in a 10-mm²patch, andeach needle had a side length of 400 μm at the base, a side length of 5μm at the tip, and a height of 800 μm (FIG. 3g, 3h ). The needle wasdesigned to have a triple-layered structure consisting of GSA, α-amyloseand crosslinked hyaluronic acid matrix using alternating deposition. Themechanical strength of MN was determined as 0.18 N/needle, which wassufficient for skin penetration without breaking (FIG. 11) (S. P.Sullivan et al. Nature Medicine 2010, 16, 915). A fluorescence viewdepicted the representative integration of MN patch with the pancreaticβ-cells capsules (FIG. 3i ). GSAs were well distributed in tip region ofthe MNs and the cell-embedded capsules were positioned on the back ofthe MN patch.

The GSIS of L-S GRS was examined through the microfluidics (FIG. 3f ).The needles on the patch were incubated in an open microfluidic channelwith continuous infusion of the Krebs-Ringer buffer (KRB) with ahyperglycemic level (400 mg/dL) and a normoglycemia level (100 mg/dL)respectively. The GSIS with the high glucose level infusion displayed a3-fold increase compared to the low glucose one (FIG. 3c ). This wasattributed to the hyperglycemic flow, which quickly promoted thedissociation of GSA; and the subsequent hydrolysis of α-amylose led toan amplified, sufficient glucose level signal for triggering thesecretion of insulin from the β-cells capsules.

To investigate the in vivo efficacy of the glucose-responsive MN device,STZ-induced type 1 diabetic mice were subjected to transcutaneousadministration of a variety of MNs samples: empty MNs without GRS (w/oGRS), MNs integrated with only L-GRS (L-GRS), MNs integrated with onlyS-GRS (S-GRS), MNs integrated with L-S-GRS (L-S GRS), MNs integratedwith L-S-GRS but without GOx in S-GRS (L-S GRS (w/o GOx)), and MNsintegrated with L-S-GRS but without α-amylose in S-GRS (L-S GRS (w/oAM)). Each MN patch was administered by a homemade applicator with5N/patch to ensure the uniform penetration and was immobilized on theskin by topical skin adhesive. The excised skin tissue clearly showedthe visible sites of needle insertion (FIG. 4a , top) and thehematoxylin and eosin (H&E)-stained cross-section image indicated thatMNs could penetrate to a depth of approximately 200 μm to the epidermis(FIG. 4a , bottom), which allowed the GSA to be exposed to interstitialfluid in real-time (S. P. Sullivan et al. Nature Medicine 2010, 16,915).

The BGLs of treated mice in each group were monitored over time. Asshown in FIG. 4b , the BGLs in mice treated with MN patch integratedwith L-S GRS quickly declined to nearly 200 mg/dL within two hours andmaintained in a significantly reduced level for 6 h without peaks ofhyperglycemic or hypoglycemic states. In contrast, without the completeS-GRS (L-GRS group) or just lacking the responsive element-GOx (L-S GRS(w/o GOx) group) or amplifying element-AM (L-S GRS (w/o AM) group), theBGLs only decreased in the first hour, which could be explained by thediffusion of residual amounts of insulin detained in the hydrogel.Afterwards, the insulin secretion of β-cells maintained at the basallevel and the BGLs of mice reverted to the hyperglycemic state. In theabsence of β-cell capsules, the groups treated with MNs integrated withonly S-GRS (S-GRS) or empty MN (w/o GRS) groups displayed no noticeabledecline in BGLs as expected. The temporarily elevated BGLs in S-GRSgroup could be attributed to the induced hydrolysis of α-amylose and thehost glucose clearance (FIG. 12).

To assess whether the MN patch could modulate the BGLs without causingpotential risks of hypoglycemia, a group of STZ-induced mice weresubjected to the MN patch replacement administration. The second MNpatch treatment 6 hour post the first administration did not secreteexcess insulin in absence of hyperglycemia trigger, which could avoidthe hypoglycemia risk. Moreover, the additional MN patch was able toprolong the treatment efficiency in response to the elevated BGLscompared to the control (FIG. 4c ). The study on the healthy micetreated with MN patches integrated with L-S GRS and empty MN as controldemonstrated that the device did not cause hypoglycemia (FIG. 4d ).Insignificant insulin release from the L-S GRS still maintained the BGLsof mice in a normal range. A glucose tolerance test demonstrated thetight glucose regulation capability on diabetic mice (J. Yu et al.Proceedings of the National Academy of Sciences 2015, 112, 8260; D.H.-C. Chou et al. Proceedings of the National Academy of Sciences 2015,112, 2401). At 2 hours after administration of the L-S GRS, the diabeticmice were treated with an intraperitoneal (i.p.) glucose injection. BGLsof diabetic mice showed a 100 mg/dL increase and rapid decline toinitial BGLs within 60 minutes (FIG. 4e ). The area under the curvebetween 0 and 120 minutes was calculated to indicate the MN maintenanceof glucose homeostasis. Significant difference was observed between MNgroup and the control group 2 h post glucose challenge (FIG. 4f ).

To assess the biocompatibility of the GSA-loaded MN patch, thecytotoxicity of dissolved microneedles toward β-cells was evaluated byMTT assay (FIG. 13). The MNs and corresponding dissolved products didnot show significant decrease of cell viability with the studiedconcentrations. The skin treated by the MN patch could rapidly recoverwithin 8 hours after MN removal and the H&E stained skin section of theinjection site presented no obvious inflammation (FIG. 14) (W. Yuan etal. Drug Design, Development and Therapy 2013, 945).

Currently, the biocompatibility and safety issues significantly hamperthe clinical applications of pancreatic islet cells transplantation (O.Veiseh et a. Nature Reviews Drug Discovery 2014, 14, 45; S. Mitragotriet al. ACS Nano 2015, 9, 6644; K. M. Bratlie et al. Advanced HealthcareMaterials 2012, 1, 267). Instead of utilizing traditional administrationmethods and relying on an invasive procedure, a microneedle patch-basedstrategy was developed to control the insulin secretion from externallypositioned pancreatic β-cells, triggered by the internal hyperglycemicstate. Importantly, for the first time, a synthetic amplifier wasincorporated to quickly amplify the physiological signal, in this case“glucose level”, for effective transport of the signal and sufficientstimulation of insulin secretion from the β-cells. The results of serialtreatments in vivo showed the potency of the MN patches in tight glucoseregulation for a prolonged period. This method circumvents thechallenging issues for pancreatic cells therapy associated with immuneresponse and long-term efficacy. This effective administration periodcan be further extended by optimizing the density and viability of cellsas well as the physicochemical properties of matrix material fortransporting glucose and insulin. In one example, freshly-preparedpatches with pig islets or stem cell-differentiated human pancreaticcells can be delivered to patients every 1, 2, 3, 4, 5, 6, 7, or moredays for ease of administration. Disclosed are synthetic amplifiers forenhancing efficacy of physiological signal-responsive drug deliverysystems when the original bio-signal is insufficient for triggeringresponsiveness.

Methods Used in Example 1 Materials

All chemicals unless mentioned were purchased from Sigma-Aldrich. Sodiumhyaluronic acid (HA, the molecular weight of 300 kDa) was purchased fromFreda Biochem Co., Ltd. (Shandong, China), custom synthetic peptide(CGRGDS, MW 594.31) was purchased from Celtek Bioscience, LLC.(Franklin, Tenn.), anti-insulin antibody (ab181547) and goat anti-rabbitIgG H&L (FITC) (ab6717) were purchased from Abcam, skin affix surgicaladhesive was purchased from Medline Industries, Inc.

Synthesis and Characterizations of Hypoxia-Sensitive Hyaluronic Acid(HS-HA)

Hypoxia-sensitive hyaluronic acid (HS-HA) was synthesized by chemicallyconjugating 6-(2-nitroimidazole) hexylamine through amide formation.First, 6-(2-nitroimidazole) hexylamine was synthesized to react with thecarboxylic acids groups of HA. In brief, NI (0.15 g, 1.3 mmol) wasdissolved in DMF, to which K₂CO₃ (0.28 g, 2.0 mmol) was added. Then6-(Boc-amino) hexyl bromide (0.39 g, 1.4 mmol) was added dropwise intothe solution and stirred at 80° C. for 4 hours. The solid impuritieswere separated from the reaction mixture through filtration and washedwith methanol. The solid product was obtained from the residual solventby evaporation, which was suspended in deionized (DI) water and thenextracted with ethyl acetate. The organic layer was collected andconcentrated with sodium sulfate. The product was re-dissolved inmethanol on the ice. 5 mL of 1.25 M HCl in methanol was added to thesolution and stirred for 24 h at room temperature (RT). Afterward, thesolvent was removed using rotary evaporator to obtain theamine-functionalized NI. Second, 6-(2-nitroimidazole) hexylamine wasconjugated to HA in the presence of1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) andN-hydroxysuccinimide (NHS). Briefly, 0.24 g of HA (molecular weight:˜300 kDa) was dissolved in water, to which EDC (0.56 g, 3.4 mmol) andNHS (0.39 g, 3.4 mmol) were added and stirred for 15 minutes at roomtemperature. Later 6-(2-nitroimidazole) hexylamine (0.18 g, 0.85 mmol)in DMF was added to the mixture and reacted at room temperature for 24hours. The dialysis was performed thoroughly against a 1:1 mixture of DIwater and methanol for 24h and DI water for 48 hours. Then, HS-HA wasobtained by lyophilization and characterized by ¹H NMR (Varian Gemini2300). The graft degree is determined as 20% by UV-Vis absorbance.

6-(2-nitroimidazole) hexylamine: ¹H NMR (DMSO-d₆, 300 MHz, δ ppm):1.30-1.78 (m, 8H, NH₂CH₂(CH₂)₄), 2.73 (s, 2H, NH₂CH₂), 4.38 (s, 2H,NCH₂), 7.19 (s, 1H), 7.87 (s, 1H). HS-HA: ¹H NMR (D₂O, 300 MHz, δ ppm):1.88-2.40 (m, 8H, NH₂CH₂(CH₂)₄), 2.87-3.19 (m, 4H, NH₂CH₂, NCH₂), 7.19(s, 1H), 7.48 (s, 1H).

Preparation and Characterization of Methacylated Hyaluronic Acid (m-HA)

Hyaluronic acid (HA) was modified with double bond by reacting with themethacrylic anhydride (MA). Two grams of HA was dissolved in 100 mL ofdistilled (DI) water in cold room overnight, followed by the dropwiseaddition of 1.6 mL of methacrylic anhydride (MA). The reaction solutionwas maintained between pH 8-9 by adding 5 N NaOH and continuouslystirred at 4° C. for 24 hours. Subsequently, m-HA was precipitated inacetone, washed with ethanol 3 times and then dissolved in DI water.After dialysis against DI water for 48 hours, the purified m-HA wasobtained with a yield of 87.5% by lyophilization and characterized by ¹HNMR (Varian Gemini 2300). The degree of modification (DM) was calculatedto be about 15% by comparing the ratio of the areas under the protonpeaks at 5.74 and 6.17 ppm (methacrylate protons) to the peak at 1.99ppm (N-acetyl glucosamine of HA) after performing a standarddeconvolution algorithm to separate closely spaced peaks.

m-HA: ¹H NMR (D₂O, 300 MHz, δ ppm): 1.85-1.96 (m, 3H, CH₂═C(CH₃)CO),1.99 (s, 3H, NHCOCH₃), 5.74 (s, 1H, CH¹H²═C(CH₃)CO), 6.17 (s, 1H,CH¹H²═C(CH₃)CO).

Rheological experiments of the m-HA hydrogel were conducted at 37° C.using a TA Instruments AR-2000 stress controlled rheometer withsandpaper covered parallel-plate (25 mm). The 2 wt % m-HA hydrogel inDulbecco's Modified Eagle Medium (DMEM) were crosslinked by in situphotopolymerization with N,N′-methylenebisacrylamide (MBA 2%, w %) andphotoinitiator (Irgacure 2959; 0.05%, wt %) via UV irradiation(wavelength: 365 nm, intensity: 9 W/cm²) for 1 minute. Experiments wereperformed within the linear viscoelastic regime at 0.5 Pa geometrieswith a 2 mm gap. Measurements were performed at least thrice to ensurereproducibility within ±10%.

Preparation and characterization of GSA GSA was prepared byself-assembly in aqueous solution. Briefly, 20 mg of amphiphilic HS-HAwas dissolved in water/methanol (2:1 vol/vol), followed by addition of 1mg of GOx from Aspergillus niger (200 U/mg, 160 kDa/7 nm), 3 mgα-amylase from Bacillus licheniformis (500-1,500 U/mg, 51 kDa/2.43 nm)and 6 mg of amyloglucosidase from Aspergillus niger (≥300 U/mL, 75kDa/3.35 nm). The mixture was stirred at 4° C. for 2 hours. Then, themethanol was removed by dialysis against deionized water for 1 day. Theresulting GSA suspension was further filtered by a centrifugal filter(100 kDa molecular mass cutoff, Millipore) at pH 7.4 to remove theunloaded enzymes by centrifugation at 14,000×g for 10 min. The final GSAsuspension was stored at 4° C. for further study. For the fluorescenceGSA, 0.01 wt % Rhodamine B were added to the enzymes solution during theGSA preparation. The loading capacity (LC) and encapsulation efficiency(EE) of enzymes-encapsulated vesicles were determined by measuring theamount of non-encapsulated enzymes through BCA (bicinchoninic acid)protein assay and using enzyme-free vesicles as basic correction. LC andEE were calculated as LC=(A−B)/C, EE=(A−B)/A, where A was expectedencapsulated amount of enzymes, B was the free amount of enzymes in thecollection solution, and C was the total weight of the vesicles. Thezeta-potential and size distribution were measured on the Zetasizer(Nano ZS; Malvern). The TEM images of GSA were obtained on a JEOL 2000FXTEM instrument.

Oxygen Consumption Rate Assay

Oxygen consumption rate (OCR) was determined by using MitoXpress (CaymanChemical) according to the manufacturer's protocol. Briefly, 200 μL 5mg/mL GSA solution with 0, 100 or 400 mg/dL glucose containing 10 μL ofMitoXpress probe was placed in a 96-well plate, and the plate wasmeasured on a microplate reader at the excitation/emission wavelength of380/650 nm at 37° C. Each sample well was measured repetitively every 5minutes, by taking two intensity readings at delay times of 30 us and 70us and gate time of 30 μs. Obtained TR-F intensity signals for eachsample well were converted into phosphorescence lifetime (μs) τ valuesas follows: τ=(70-30)/ln(F1/F2), where F1 and F2 are the TR-F intensitysignals at delay times 70 μs and 30 μs. The resulting increasinglifetime ti reflects the oxygen concentration in each sample.

In Vitro Glucose-Responsive Studies of the GSA

To evaluate the glucose-responsive capability of GSA, GSA were incubatedin 500 μL of PBS buffer (NaCl, 137 mM; KCl, 2.7 mM; Na₂HPO₄, 10 mM;KH₂PO₄, 2 mM; pH 7.4) with 100 μM NADPH and 5 μg/mL cytochrome creductase. The α-amylose was first washed with ethanol and then added tothe PBS solution (10 mg/mL). Various amounts of 45% glucose solution(Corning) were added to reach a final solution of various glucoseconcentration (0 mg/dL, 100 mg/dL, and 400 mg/dL). The mixture wasincubated at 37° C. in a container with an oxygen concentration of 21%by regulation of a mass flow meter. At predetermined time intervals, thereleased enzymes were separated from GSA suspension by a centrifugation(10 kDa molecular mass cutoff, Millipore) at 14,000×g for 10 minutes.The concentration of residual enzymes encapsulated in GSA and thereleased enzymes separated from the GSA was examined using a CoomassiePlus protein assay. The A595 was detected on an Infinite 200 PROmultimode plate reader (Tecan Group Ltd.), and the enzyme content wascalibrated with the standard curve of the enzyme solutions. The glucosewere further separated by a centrifugal filter (10 kDa molecular masscutoff, Millipore) at pH 7.4 by centrifugation at 14,000×g for 10minutes. The glucose concentration was determined by Glucose (GO) assay.Briefly, the samples were diluted properly, and then added with assayreagent for 30 minutes at 37° C. The reactions were stopped at 30-60second intervals by adding 2.0 mL of 12 N H₂SO₄ into each tube. Theabsorbance was measured against the reagent blank at 540 nm. The glucoseconcentration were calculated from the standard curve. For plotting theUV-Vis absorption of GSA solution, the intensity was measured at an A of330 nm at the set time. The far-UV CD spectra of the native and releasedAA and GA from GSA (1 μM) were analyzed by the CD spectrometer (Aviv).

Fabrication of GSA-loaded MNs

The microneedle fabrication was carried out using 10 uniform siliconemolds from Blueacre Technology Ltd. Each mold is characterized with20×20 arrays of microneedle pyramid cavities machined by laser ablation.The needle cavity had a side length of 400 μm at the base, a height of800 μm and a side length of 5 μm at the tip. After plasma cleaning themolds, 50 μL GSA solution was deposited onto the mold surface. It wasthen followed by vacuum (600 mmHg) treatment for 5 min to allow the GSAsolution flow into the MN cavities and achieve desirable viscosity.Afterwards, the molds were transferred to a Hettich Universal 32Rcentrifuge for 20 minutes at 2000 rpm to compact GSA to the tips region.After the GSA layer completely dried, second layer of 0.5 mL 10 mg/mLα-amylose solution was fed to the mold and was fabricated using the sameprocess. The mold surface then was pipetted with 300 μL m-HA solution (4wt %) mixed with MBA (2 wt %) and photo initiator (0.5 wt %) followed bycombination of vacuum and centrifugation. The process was repeated 3-4times until m-HA layer was dried and no obvious bubbles arising from themold cavities under vacuum condition. For the backing of the MN patch, apiece of 4 cm×10 cm silver adhesive tape was applied around the 2 cm×2cm mold baseplate, and 3 mL HA (5 wt %) solution was added to theprepared micromold reservoir and dried at 25° C. (overnight in vacuumdesiccator). After desiccation was completed, the microneedle patch wascrosslinked through in situ polymerization under 30s UV irradiation(wavelength of 365 nm). The resulting product was carefully separatedfrom the mold and tailored to fit the homemade applicator. The final MNscan be stored in 4° C. within a sealed six well cell culture plate for aweek.

Mechanical Strength Test

The mechanical strength of a MN was measured with a stress-strain gaugeby pressing the needle against a stainless-steel plate on an MTS 30Gtensile testing machine. The initial gauge was set as 2.00 mm betweenthe MN tip and the stainless steel plate, with 10.00 N as the cellloading capacity. The speed of the top stainless-steel plate movingtoward the MN was set as 0.1 mm·s⁻¹. The failure force of MN wasrecorded as the needle began to buckle.

Cell Culture

Mouse insulinoma cell line 6 (MIN6) cells were kindly provided by Dr.Michael McManus, University of California, San Francisco. The culturemedium used was DMEM high glucose medium with fetal bovine serum (15%),penicillin/streptomycin (1%) and 2.5 uL of islet mercaptoethanol(Biorad) per 500 mL media at 37° C. and 5% CO2. Media was changed every3 days and the cells were passaged at 60% confluency. The 32-38thpassages of the MIN6 cell lines were used.

Encapsulation of Pancreatic β-Cells

An aqueous solution of 2 wt % alginate was centrifuged at 12 000 rpm toremove any impurities. The alginate (120 mg) solution was mixed with1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide(EDC)/N-hydroxysuccinimide (NHS) (50 mg/30 mg) in pH 5.0 acetic bufferfor 30 minutes to activate carbonyl groups on alginate, followed bymixing with additional 1,6-diaminohexane (60 mg) for another 4 h. Themixture was precipitated in 2-propanol (IPA) to remove unreacteddiamine. The alginate-amine derivative was reacted with the peptidesequence of CGRGDS (the weight ratio between peptide to alginate was0.02:1) that has the Cys residue of free thiol in sodium bicarbonatesolution (50 mM; pH=8.5) for 4 h at 4° C. The peptide-modified alginatewas purified via extensive dialysis with deionized water for 5 d (3 500Da molecular mass cutoff, Millipore), sterilized through a 0.22-μmfilter and lyophilized. MIN6 cells were trypsinized and suspended at(2×10⁶ cell/mL) in 2% alginate-RGD culture medium supplemented with 1%type IV collagen. The mixture was transferred into a 1 mL syringe withan attached blunt tip, 22 gauge metal needle. The syringe was placed inan electrospray system equipped with a syringe pump. The positiveelectrode of the electrospray system was connected to the needle, andthe negative electrode was connected to a metal receiving container with50 mL of 20 mM BaCl₂. The solution was sprayed at a 0.155 mL/min flowrate under a high voltage (8 kV) with a working distance of 5 cm to thereceiving container. After droplets (50-65 uL) were extruded, the innerphase was gelatinized in sterilized BaCl₂ (200 mM) solution for 5minutes. The collected capsules were then rinsed three times withsterilized NaCl solution (150 mM) before transferring to DMEM media in a12 well culture plate. Homogeneous number of ˜100 capsules werecollected and further introduced into a bulk hydrogel for eachexperimental group to permit comparison across treatments. The HAhydrogel were crosslinked by photopolymerization of m-HA in DMEM (2%, wt%, DMEM) with N,N′-methylenebisacrylamide (MBA 2%, wt %) andphotoinitiator (Irgacure 2959; 0.05%, w %) via UV irradiation(wavelength: 365 nm) for 1 minute (FIG. 15).

Characterization of Pancreatic β-Cell Capsules

To analyze the morphological characteristics of cellular clusters, thepancreatic β-cell capsules were viewed under Olympus IX70multi-parameter microscope. The average diameter of the pancreaticβ-cell capsules was fitted to an ellipse and analyzed by adjusting theimages using the particle analyzing method of the ImageJ software.

Qualitative cell viability was visualized by the LIVE/DEADViability/Cytotoxicity Kit (Invitrogen). Cell capsules were incubated inPBS (NaCl, 137 mM; KCl, 2.7 mM; Na₂HPO₄, 10 mM; KH₂PO₄, 2 mM; pH 7.4)with 4 μM calcium AM and 8 μM ethidium homodimer-1 for 1 hour. Capsuleswere then rinsed in PBS to remove the excess staining solution and fixedwith 2% paraformaldehyde for 15 minutes. The capsules were placed on aglass slide and imaged via Leica DM5500B Fluorescent Microscope equippedwith a digital camera and the compatible LAS-AF software. Viability wasquantified by counting the number of live (green) (excitation, 494 nm;emission, 517 nm) and dead (red) (excitation, 528 nm; emission, 617 nm)cells.

Characterization of Glucose-Responsive System (GRS)

The morphology of the MN patch was characterized using SEM. Themicroneedles along with their bases were attached to an SEM sample stubusing a double-stick carbon tab. The samples were coated with a 7 nmthick gold-palladium layer using an EmTech Turbo EM sputter coater.Imaging was carried out on a FBI Verios 460L field-emission scanningelectron microscope (FESEM) at the Analytical Instrumentation Facility,North Carolina State University. Calcium AM-stained pancreatic β-cellcapsules were positioned on the back of a MN patch loaded withrhodamine-labeled GSA. The fluorescence image was taken from side viewof the L-S GRS by Olympus IX70 multi-parameter fluorescence microscope.

Glucose Stimulated Insulin Secretion

Functional assessment of encapsulated cells was tested via staticglucose-stimulated insulin secretion (GSIS) assay. 2-3 days before theexperiment, 2×10⁵ MIN6 cells were seeded per 96 well plate (CorningCostar). Similar to the 2D cell culture, same amount of cells in 3Dcapsules were cultured in 96 well plate (10/each well) 1 days prior toglucose treatment. The Kerbs-Ringer (KRB) buffer (128 mm NaCl, 4.8 mMKCl, 1.2 mm KH₂PO₄, 1.2 mm MgSO₄, 2.5 mm CaCl₂, 10 mM HEPES, 0.1% (w/v))supplemented with 0.1% BSA was prepared beforehand. The cells wereincubated for 2h in KRB buffer BSA, at 37° C., 5% CO₂, and thenincubated for 45 min with 100 mg/dL or 400 mg/dL glucose in the samecondition. The amount of insulin secreted by the cells was quantifiedusing a mouse insulin ELISA kit (Alpco.). The amount of insulinsecretion in 400 mg/dL glucose containing KRB was normalized to insulinsecreted in 100 mg/dL glucose containing KRB and expressed as insulinsecretion index.

Immunofluorescence Imaging

Six hours after encapsulation, the cells were fixed in 4%paraformaldehyde at 4° C. and then embedded in OCT compound (SakuraFinetek) and flash-frozen in an isopentane bath on dry ice. The frozencell capsules were sectioned (5-μm thickness), mounted on microscopeslides, and stored at −80° C. For staining of insulin withimmunofluorescence, the slides were washed twice, permeabilized for 30min using a 0.1% Triton X100 solution, and subsequently blocked for 1hour using a 1% bovine serum albumin (BSA) solution. After blocking,primary rabbit monoclonal to insulin antibody (abcam 181547) at 1/200dilution was applied overnight at 4° C., followed by washing andincubation with secondary antibody goat anti-rabbit IgG (Alexa Fluor®488) (abcam 150077) at 1/400 dilution (green). Slides were washedthrice, applied with cell permeant dye DAPI to stain the cell nucleusand covered with coverslips. Samples were imaged using the Olympus IX70multi-parameter fluorescence microscope and processed using ImageJsoftware.

In Vitro Glucose-Responsive Studies of the GRS

Release of insulin from the MNs was performed using a microfluidicsdevice as a lab-on-chip simulation of blood circulation system. Todynamically evaluate the glucose-responsive capability, the MN patch wasplaced in the open center of the microfluidic channel while the releasemedia flowing through the needle tips (pH 7.4 KRB with various glucoseconcentration). The infusion and withdraw rates were set at 50 μL/min ontwo separate syringe pumps (Harvard Apparatus PHD 2000, Holliston,Mass.). The insulin release rate was quantified using a mouse insulinELISA kit (Alpco.). The insulin content was measured at 450 nm on theInfinite 200 PRO multimode plate reader (Tecan Group Ltd., Switzerland),and calibrated with an insulin standard curve.

In Vivo Studies Using STZ-Induced Diabetic Mice

The in vivo efficacy of MN patches for diabetes treatment was evaluatedon STZ-induced adult diabetic mice (male C57B6, Jackson Lab, U.S.A.).The animal study protocol was approved by the Institutional Animal Careand Use Committee at North Carolina State and University and Universityof North Carolina at Chapel Hill. The plasma-equivalent glucose wasmeasured from tail vein blood samples (˜3 μL) of mice using the ClarityGL2Plus glucose meter (Clarity Diagnostics, Boca Raton, Fla.). TheirBGLs were monitored for two days before administration, and all micewere fasted overnight before administration. Five mice for each groupwere selected to be subcutaneously treated with MNs containing empty MNswithout GRS (w/o GRS), MNs integrated with only L-GRS (L-GRS), MNsintegrated with only S-GRS (S-GRS), MNs integrated with L-S-GRS (L-SGRS), MNs integrated with L-S-GRS but without GOx in S-GRS (L-S GRS (w/oGOx)), and MNs integrated with L-S-GRS but without α-amylose in S-GRS(L-S GRS (w/o AM)). MN patches were applied on the dorsum skin by ahomemade applicator with 5 N/patch for 10 min. The patch was fixed onthe skin for sustained release using skin affix surgical adhesive. A 12mm×12 mm×5 mm customized PDMS mold was covered on the patch. Inside thePDMS mold, pancreatic β-cells capsules were embedded in a crosslinkedm-HA hydrogel made from DMEM for nutrients supply. For the MN (S-GRS),MN (w/o GRS) groups, no cells were incorporated into the devices. TheBGLs of administrated mice in each group were then continuouslymonitored (at 5, 15, 30, and 60 minutes, and once per hour afterward). Aglucose tolerance test was conducted to confirm the in vivo glucoseresponsiveness of MNs 2 h post-administration of MNs. Briefly, mice werefasted overnight and administered with L-S GRS. When the mice glucoselevels reached the lowest at 2 hours post administration, mice weregiven 1.5 g glucose/kg (45% sterile glucose solution, corning cellgro)via i.p. injection. The glucose tolerance tests on healthy mice wasconducted as controls. Blood was drawn from the tail vein, and glucoselevels were measured using a glucometer at 0, 10, 20, 30, 40, 60, 90,and 120 minutes after glucose administration. Similarly, to assess therisks of hypoglycemia, two groups of healthy mice were administered withMN (L-S GRS) or MN (w/o GRS), but were not subjected to a glucosechallenge.

Biocompatibility Study

Cell proliferation analysis was performed using a colorimetric methylthiazolyl tetrazolium (MTT) assay. MIN6 cells were seeded into 96-wellplates at a density of 5,000 cells per well and cultivated in 100 μL ofDMEM. The plates were then incubated in 5% CO₂ and at 37° C. for 12hours to reach 70-80% confluency before addition of serial dilutions ofthe dissolved MN solution. After incubation for 24 hours, the cells werewashed with KRB solution and incubated with 100 μL fresh FBS free DMEMand fresh prepared 20 μL3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution(MTT solution, 5 mg/mL). The plates were incubated for an additional 4hours. After that, the culture media was carefully removed and thenfollowed by the additional of 150 μL dimethyl sulfoxide (DMSO). Theabsorbance of the plates was read at 590 nm and a reference wavelengthof 620 nm using a microplate reader (Infinite M200 Pro, Tecan,Morrisville, N.C., USA) within 10 minutes.

To evaluate the biocompatibility of MN patch in the mouse model. On day2 post-administration, mice were euthanized by CO₂ asphyxiation and thesurrounding tissues were excised. Mice with PBS administration were usedas negative control. The tissues were fixed in 10% formalin and thenembedded in paraffin, cut into 50-μm sections, and stained using H&E forhistological analysis.

Statistical Analyses

Data are presented as means±SD. Statistical analysis was performed usingthe student t test or an ANOVA test. With a P≤0.05, the differencebetween experimental groups and control groups were consideredstatistically significant.

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1. A method of treating a disease in a subject in need thereof, themethod comprising: a) providing a microneedle patch to the subject,wherein the microneedle patch comprises: i) a plurality of microneedleseach having a base end and a tip; ii) a substrate to which the base endsof the microneedles are attached or integrated; iii) at least onereservoir which is in connection with the base ends of the microneedlesarray, wherein the reservoir comprises an agent delivery system, whereinthe agent delivery system comprises an agent to be transported acrossthe biological barrier, or a means for producing an agent to betransported across the biological barrier, and a means for detecting aphysiological signal from the recipient wherein said means for producingan agent to be transported across the biological barrier and said meansfor detecting a physiological signal both comprise cells; and iv) asignal amplifier system integrated within the microneedles, wherein thesignal amplifier system comprises a component capable of amplifying thephysiological signal from the recipient, wherein the means for producingan agent to be transported across the biological barrier produces anagent, or changes the amount of agent produced, based on an amplifiedsignal from the signal amplifier system; b) inserting the microneedlesinto the biological barrier; and c) delivering the agent through themicroneedles and into the biological barrier, wherein the amount orvolume of agent delivered is determined by a signal received from thesignal amplifier system.
 2. The method of claim 1, wherein the cells arepancreatic β cells or stem cell-differentiated human pancreatic cells.3. The method of claim 1, wherein the reservoir is composed of amaterial matrix that can be used to deliver cell products.
 4. The methodof claim 3, wherein the reservoir comprises an alginate microgel.
 5. Themethod of claim 1, wherein the reservoir comprises a therapeutic,prophylactic, or diagnostic agent.
 6. The method of claim 1 wherein theagent is selected from the group consisting of peptides, proteins,carbohydrates, nucleic acid molecules, lipids, organic molecules,biologically active inorganic molecules, and combinations thereof. 7.The method of claim 6, wherein the agent is insulin.
 8. The method ofclaim 1, wherein the signal amplifier system magnifies a signal, therebyaltering the volume or amount of agent to be transported based on thestrength of the signal present in the subject.
 9. The method of claim 8,wherein the physiological signal is a biological substance in thesubject.
 10. The method of claim 1, wherein the agent delivery systemcomprises a feedback component, such that volume or amount of the agentto be transported across the biological barrier can be altered based onthe physiological signal.
 11. The method of claim 10, wherein thephysiological signal from the recipient is insufficient to be detectedby the feedback component without amplification from the signalamplifier system.
 12. The method of claim 11, wherein the physiologicalresponse in the subject comprises physiological environment factors,including pH, temperature, blood glucose levels and other biomarkers.13. The method of claim 12, wherein the signal is glucose.
 14. Themethod of claim 1, wherein the signal amplifier system comprisesself-assembled polymeric nanosized vesicles.
 15. The method of claim 14,wherein the glucose signal amplifier comprises glucose oxidase,α-amylase, and glucoamylase.
 16. The method of claim 1, wherein thesignal amplifier system is in the tip of the microneedle.
 17. The methodof claim 1, wherein the subject has been diagnosed with diabetes.